The present invention relates to a method for the preparation of a conjugate comprising a first and a second polypeptide, said method comprising the steps of (a) incubating said first polypeptide in the presence of a heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer; (b) removing excess heterobifunctional crosslinker; and (c) incubating the reaction product of step (b) with said second polypeptide, wherein said second polypeptide comprises at least one sulfhydryl group. Furthermore, the present invention relates to a conjugate obtainable by the method of the present invention. Also described is a pharmaceutical composition comprising the conjugate of the present invention and, optionally, a pharmaceutically acceptable carrier and/or diluent, and the use of the conjugate for the preparation of a pharmaceutical composition for preventing and/or treating an allergic disease or an autoimmune disease.
Immunologic tolerance may be defined as a state of antigen-specific unresponsiveness induced by preexposure to an antigen. If the antigen is an allergen, the immune response is defined as allergy, an adverse reaction with an immunologic basis mediated by IgE immunoglobulin (Sampson (1986), J. Allergy Clin. Immunol. 78:212-219). The immune system may also be a cause of disease or other undesirable consequences, when the principle of self/non-self recognition breaks down and the body""s own components are recognized as non-self (autoantigens) in which case autoimmune diseases can ensue.
Interest in immunologic tolerance, discovered by Medawar almost half a century ago (Billingham et al. (1953), Nature 172:603-606), has increased for two main reasons: (1) Several of its mechanisms, such as clonal deletion (Kappler et al. (1987), Cell 49:273-280), anergy (Jenkins and Schwartz (1987), J. Exp. Med. 165:302-319), and regulatory T cells (Gershon and Kondo (1971), Immunology 21:903-914) have been uncovered. (2) Both systemic and oral tolerance (Cremer et al. (1983), J. Immunol. 131:2995-3000; Weiner et al. (1994), Ann. Rev. Immunol. 12:809-837) can be induced to, it is hoped, prevent either autoimmune or allergic diseases. For example, several strategies have been used in trying to prevent allergy, including administration of modified allergen (Lee and Sehon (1977), Nature 267:618-649), allergen linked to nonimmunogenic carriers (Katz et al. (1971), J. Exp. Med. 134:201-203), single peptides (Muckerheide et al. (1977), J. Immunol. 119:1340-1345), or an allergen-antibody complex (Machiels et al. (1990), J. Clin. Invest. 85:1024-1035).
It is known that antigen presentation can influence the type of immune response. Not only haptens (Borel (1989), in xe2x80x9cConcepts in Immunopathologyxe2x80x9d, Cruse and Lewis (Eds.), 7:145-161, Karger, Basel; Sehon (1982), Prog. Allergy 32:161-202) but also proteins covalently linked to a carrier molecule naturally tolerated by the host, such as isologous immunoglobulin, can induce unresponsiveness to these proteins (Filion et al. (1980), Cell Immunol. 54:115-128; Borel and Borel (1990), J. Immunol. Methods 126:159-168).
However, although the above strategies proved to be partially successful, there is still a need for allergen and/or auto-antigen comprising conjugates with improved therapeutic properties.
Thus, the technical problem underlying the present invention was to provide a method for the production of such allergen and/or auto-antigen comprising conjugates.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a method for the preparation of a conjugate comprising a first and a second polypeptide, said method comprising the steps of:
(a) incubating said first polypeptide in the presence of a heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer;
(b) removing excess heterobifunctional crosslinker; and
(c) incubating the reaction product of step (b) with said second polypeptide, wherein said second polypeptide comprises at least one sulfhydryl group.
It is envisaged in accordance with the present invention that the polyethylene oxide spacer may consist of from 1 to 10 monomer units. Preferably, the spacer consists of from 2 to 5 monomer units.
Unexpectedly, it has been found in accordance with the present invention that due to the crosslinker used conjugates prepared by the method of the present invention show superior features as compared to conjugates prepared by prior art methods. For example, conjugates of ragweed derived antigen (Amba-l) and mouse IgG were prepared by the method of the present invention and a method using as crosslinker N-(xcex3-maleimidobutyroxy)sulfosuccimimide ester (sulfo-GMBS) a preferred prior art crosslinker (see Example 1, infra). The effects of both conjugates in the treatment of an allergic reaction to ragweed derived antigen were investigated. Surprisingly, it was found that the conjugate of the present invention leads to a significant reduction in airway hyperresponsiveness whereas the prior art conjugate has almost no effect when compared to sensitized animals (see Example 5 and FIG. 3). Furthermore, the conjugate of the present invention virtually eliminated eosinophils in the bronchoalveolar lavage fluid (BALF). This phenomenon was accompanied by a total suppression of specific anti-Amba-l IgE. In contrast, the prior art conjugate only moderately reduced eosinophils, and specific anti-Amba-l IgEs were only partially suppressed (see Example 5 and FIG. 4). These results clearly demonstrate that due to the crosslinker used the method of the present invention allows the person skilled in the art to produce conjugates with advantageous immunological properties which render these conjugates for instance suitable for the downregulation of the inflammatory and immunologic reactions of an allergic response.
In a preferred embodiment of the present invention, said at least one sulfhydryl group of said second polypeptide is introduced by:
(i) incubating said second polypeptide in the presence of N-succinimidyl-S-acetylthioacetate (SATA);
(ii) removing excess SATA;
(iii) incubating the reaction product of step (ii) in the presence of hydroxylamine; and
(iv) removing excess hydroxylamine and acetylated hydroxylamine.
This embodiment is of particular importance in cases where said second polypeptide does not comprise at least one endogenous sulfhydryl group that is, for example, provided by a cysteine residue in the amino acid sequence, and which is suitable for crosslinking said second polypeptide with said first polypeptide via a disulfide bond. Sulfhydryl groups that are not suitable for crosslinking may be, for example, sulfhydryl groups that are not readily accessible for the sulfhydryl group of said first polypeptide due to the three dimensional conformation of said second polypeptide, or that are not available due to inter- or intramolecular disulfide bonds. Whether said second polypeptide comprises one or more sulfhydryl groups that allow effective crosslinking to occur or whether sulfhydryl groups have to be introduced into said second polypeptide can be determined by the person skilled in the art without further ado. For example, two polypeptides may be crosslinked by the method of the present invention and the quality and quantity of the obtained conjugate may be analyzed by SDS polyacrylamide gel electrophoresis under non-reducing conditions and immunoblotting with an antibody specific for one of the two crosslinked polypeptides. Preferably, the identity of the conjugate is verified with a second antibody specific for the second of the two polypeptides. For a detailed description of the above outlined experiment, reference is made to Example 1. If the results show that the quality and quantity of the conjugate is not satisfactory as compared to a reference conjugate like, for example, the IgG/Amba-l conjugate produced in Example 1, sulfhydryl groups may be introduced as described above.
In another preferred embodiment, said heterobifunctional crosslinker comprising an N-hydroxylsuccinimide ester group and a maleimide group linked via a polyethylene oxide spacer has the formula: 
In a further preferred embodiment of the method of the present invention, the heterobifunctional crosslinker is used in step (a) in a 5- to 50-fold higher molar concentration than said first polypeptide.
In yet another preferred embodiment, steps (a) and (c) are performed in a temperature range from 20xc2x0 C. to 37xc2x0 C.
In a still further preferred embodiment, steps (a) and (c) are performed in a time range from 30 min to 120 min.
In another preferred embodiment of the method of the present invention, steps (a) and (c) are performed in a pH range from 7.0 to 9.0.
In a further preferred embodiment, the molar ratio of said first and second polypeptide is between 1:1 and 1:10.
In a more preferred embodiment of the method of the present invention, steps (a) and (c) are performed in a buffer comprising 0.15 M borate, pH 8.0 at 37xc2x0 C. for 30 min, the heterobifunctional crosslinker is used in a 30-fold higher molar concentration than said first polypeptide, and the molar ratio of said first and second polypeptide is 1:10.
These conditions could be shown in accordance with the present invention to result in conjugates that are most effective and beneficial if used, for example, to counteract allergic reactions.
In another preferred embodiment, the method of the present invention further comprises the step of removing aggregated conjugate and providing the conjugate in monomeric form.
Unexpectedly, this step even further improves the properties of the conjugates of the present invention.
In a still further preferred embodiment, said first polypeptide is an immunoglobulin or a structurally equivalent fragment thereof.
As discussed above, the method of the present invention may be, inter alia, used to produce conjugates that, if comprising an allergen, may be used as a xe2x80x9ctolerogenxe2x80x9d, i.e. as a conjugate capable of inducing immunologic tolerance in a subject allergic to the allergen it comprises. Without wanting to be bound to a specific scientific theory, it is envisaged that these tolerogenic properties are conferred to the conjugate by a molecule that is also comprised by the conjugate, and is recognized by the subject as xe2x80x9cselfxe2x80x9d like, for example, an isologous or autologous immunoglobulin. Accordingly, the term xe2x80x9cstructurally equivalent fragmentxe2x80x9d as used in accordance with the present invention denotes fragments that show the same immunological properties as the entire immunoglobulin, i.e. that do not induce an immune response in the subject. Such a fragment may be, for instance, the Fc portion of an immunoglobulin.
In a more preferred embodiment, said immunoglobulin or structurally equivalent fragment thereof is an immunoglobulin G or a structurally equivalent fragment thereof.
In yet another preferred embodiment, said second polypeptide is an allergen, an autoantigen or an immunologically equivalent fragment of said allergen or autoantigen.
xe2x80x9cImmunologically equivalent fragmentxe2x80x9d as used in accordance with the present invention denotes a fragment of an allergen or autoantigen that is capable of inducing the same immune response in a subject as the corresponding allergen or autoantigen, i.e. the same allergic or autoantigenic reaction.
Since it could be shown that conjugates comprising an immunoglobulin and, for example, an allergen induce unresponsiveness in a subject allergic to said allergen, it is envisaged in accordance with the present invention that conjugates comprising an immunoglobulin and, for example, an autoantigen can be used to induce unresponsiveness to said autoantigen and, thus, be used prophylactically in subjects suspected to have a predisposition for the development of the corresponding autoimmune disease.
In a more preferred embodiment said allergen is derived from ragweed, birch pollen, peanut, house dust mite, animal danders, mould, or is tropomyosin or an immunologically equivalent fragment thereof.
In another more preferred embodiment, said autoantigen is acetylcholine receptor, insulin, insulin receptor, myelin basic protein or an immunologically equivalent fragment of these proteins.
In another embodiment, the present invention relates to a conjugate obtainable by the method of the present invention.
As could be shown in accordance with the present invention, the conjugates of the invention do not only protect subjects against hypersensitivity type I reactions and, thus, the development of allergy. The conjugates of the inventions are also effective in downregulating allergic reactions and, thus, may be used to treat allergic diseases. Likewise, it is envisaged that conjugates comprising an immunoglobulin and, for example, an autoantigen may be used to ameliorate and/or treat the corresponding autoimmune disease and/or symptoms associated therewith.
The present invention also relates to a pharmaceutical composition comprising the conjugate of the present invention and, optionally, a pharmaceutically acceptable carrier and/or diluent.
Examples of suitable pharmaceutical carriers are well known in the art and include saline solutions, water, emulsions, such as oil/water emulsions, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected orally or parenterally, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration, or directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient""s size and weight, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 xcexcg. However, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 xcexcg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 xcexcg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer""s dextrose, dextrose and sodium chloride, lactated Ringer""s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer""s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Furthermore, the present invention relates to the use of the conjugate of the present invention for the preparation of a pharmaceutical composition for preventing and/or treating an allergic disease or an autoimmune disease.
In a preferred embodiment of the use of the present invention, wherein said allergic disease is an allergic reaction against an allergen derived from ragweed, birch pollen, peanut, house dust mite, animal danders, mould, or tropomyosin or an immunologically equivalent fragment thereof.
In another preferred embodiment, said autoimmune disease is Myasthenia gravis, type I Diabetes or multiple sclerosis.